The Pseudomonas aeruginosa type III secreted toxin ExoT is necessary and sufficient to induce apoptosis in epithelial cells

Abstract

SummaryType III secreted (T3SS) effectors are important virulence factors in acute infections caused by Pseudomonas aeruginosa. PA103, a well-studied human lung isolate, encodes and secretes two effectors, ExoU and ExoT. ExoU is a potent cytotoxin that causes necrotic cell death. In addition, PA103 can induce cell death in macrophages in an ExoUindependent but T3SS-dependent manner. We now demonstrate that ExoT is both necessary and sufficient to cause apoptosis in HeLa cells and that it activates the mitochondrial/cytochrome c-dependent apoptotic pathway. We further show that ExoT induction of cell death is primarily dependent on its ADP ribosyltransferase domain activity. Our data also indicate that the T3SS apparatus can cause necrotic cell death, which is effectively blocked by ExoT, suggesting that P. aeruginosa may have evolved strategies to prevent T3SS-induced necrosis.

An investigation of type 3 secretion toxins encoding-genes of Pseudomonas aeruginosa isolates in a University Hospital in Egypt

“…5,7 ExoT targets host kinases involved in focal adhesion and phagocytosis, 7 and has been associated with dissemination of disease from the lung to the liver in mice 8 and induction of apoptosis in HeLa cells. 9 ExoY is an adenylate cyclase that cleaves the intracellular cAMP in eukaryotic cells causing rounding of certain cell types. It shows little pathology in mouse pneumonia 6 , but has significant cytotoxicity in MDCK cells.…”

Pseudomonas aeruginosa exotoxin T induces potent cytotoxicity against a variety of murine and human cancer cell lines

“…ExoT was delivered into cancer cells either by P. aeruginosa bacteria or by transient transfection using pIRES2 mammalian expression vector and cytotoxicity was assessed as described previously (Shafikhani et al, 2008;Wood et al, 2013). Briefly, bacteria were grown overnight in LB media, adjusted to OD 600 0.05, corresponding to m.o.i.~10.…”

“…For transfection experiments, cells were grown as described above and transfected with Effectene (Qiagen) as per the manufacturer's protocol 1 h prior to the addition of PI and the start of time-lapse video microscopy. Time-lapse videos were assessed for cytotoxicity by determining the percentage of transfected GFP that became PI-positive, as described previously (Shafikhani & Engel, 2006;Shafikhani et al, 2008;Wood et al, 2013).…”

Determination of lethal dose (LD50) of Exotoxin A from Pseudomonasaeruginosa26A in mice histopathology

“…[22] reveal that transmission electron micrographs of mouse livers excised12 h following injection of higher doses of PEA show that the dying cells were hepatocytes. They diedby apoptosis and necrosis whereasneither apoptosis nor necrosis was seen in livers of saline-treatedmice.…”